In Vitro Model of Latent Mycobacterial Infection

ABSTRACT

A method of inducing latency in  Mycobacterium  permits preparation of an in vitro model system of latent mycobacterial infection. Latency is induced in a pure culture of  Mycobacterium  by exposing it to multiple stress conditions, including a low nutrient culture medium without glycerol, a low pH, a relatively high level of carbon dioxide and a relatively low gas phase oxygen level. An in vitro model of mycobacterial infection employs macrophages induced from THP1 cells which are then infected with  Mycobacterium . The infected macrophages are grown under hypoxic conditions to induce latency in the mycobacteria. The in vitro model of infection is useful in evaluating compounds for activity against latent mycobacteria.

RELATED APPLICATION

This application claims priority from co-pending provisional applicationSer. No. 60/914,838, which was filed on Apr. 30, 2007, and which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of infectious diseases and,more particularly, to a pure culture of Mycobacterium which exhibitslatency, including resistance to rifampicin and storage of increasedlipids, and to a method for generating such a culture.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) remains the leading cause of preventable deaths in theworld with 100 million new infections and two million deaths each year.TB is caused by Mycobacterium tuberculosis (hereinafter also referred toby the abbreviation “Mtb”), an acid-fast bacillus that is transmittedprimarily via the respiratory route. The aerosol containing the pathogenis released from people with active TB when they cough or sneeze. When aperson breathes in the pathogen it enters the alveolar macrophages via avariety of receptors. Mtb multiplies within the vacuoles in themacrophage, avoids fusion with the acidic lysosomes and eludes the hostdefenses. As the host defense system senses the multiplying pathogen andmounts its immune defense, the pathogen goes into a non-replicating,drug-resistant, latent state. The protective response by the immunesystem at the site of infection results in the formation of a granulomathat contains the infection and prevents its spread. Live bacilli havereportedly been isolated from granulomas or tubercles in the lungs ofpersons with clinically inactive tuberculosis, regarded as the latentform of TB, indicating that the organism can persist in granulomatouslesions for decades. It is estimated that one-third of the worldpopulation has latent TB. These individuals are asymptomatic latentcarriers who exhibit no signs of disease. Their risk for reactivation isestimated to be 2-23% over their life time. One study concluded that a25 year old with latent TB has a 7.3% life time risk of reactivation.The risk increases dramatically for persons coinfected with HIV, morelike 10% per year. Thus, the advent of AIDS greatly amplified the TBthreat to human health. The deadly partnership between TB and AIDS,especially with multi- and extremely drug-resistant TB, is contributingto a dramatic rise in TB cases worldwide leading to a grave situation.The emergence and spread of multi-drug resistant and extremelydrug-resistant TB is widely recognized as a major threat to publichealth.

The ability of the pathogen to go into the drug-resistant latent stateis a major road block to the eradication of TB. It is known that latentMtb persists in a non-replicating state. Antibiotics used to treatbacterial infection are usually active against growing bacteria but notagainst the dormant pathogen. Correlation between antibiotic activityand bacterial growth state in streptomycin-dependent Mtb was shownalmost 30 years ago. The antibiotic-resistance of non-growing bacteriais due to changes in bacterial metabolism or physiological state and isdescribed as phenotypic resistance. The phenotypic resistance has beenclassified into three types based on the physiological state of bacteriaas stationary phenotyopic resistance, persister phenotypic resistanceand phenotypic resistance in dormant bacteria. Mtb displaysdormancy-related phenotypic resistance which is demonstrated by theCornell mouse model. Traditionally, the phenotypic resistance isexemplified by resistance to the antibiotic Rifampicin (Rif) and isregarded as one of the hallmarks of latent TB. The mechanism ofphenotypic resistance in dormant Mtb is not clearly understood.

Development of drugs that can effectively kill dormant Mtb is of vitalimportance for the eradication of TB. If such drugs would prevent thepathogen from surviving in a drug-resistant state, a combination of suchdrugs with currently used antibiotics could drastically shorten theperiod of treatment for complete cure and lead to global eradication ofTB. For this purpose, we need to identify processes that are necessaryfor the pathogen to go into dormancy, survive under the nonreplicatingdrug-resistant state, and get reactivated when the immune system of thehost is weakened. Such steps, essential for the latent pathogen, couldoffer ideal targets for novel antilatency drugs that can eliminate thedormant pathogen. To achieve these objectives we explored thebiochemical processes that the pathogen uses to survive for such longperiods under a latent state. It has been known for many decades thatMtb in the host uses fatty acids as the major source of energy. It iswell known that glyoxylate cycle is used by organisms that live on fattyacids. In recent years the important role of isocitrate lyase, a keyenzyme uniquely used in the glyoxylate cycle, was shown to be requiredfor the persistence of Mtb in the host demonstrating the central roleplayed by fatty acid catabolism in persistence. However, the source offatty acids used by the pathogen remains unclear. We postulated that thepathogen probably stores energy as triacylglycerol (TG) as it goes intodormancy and uses this stored energy to survive the long dormant periodat very low metabolic rates as many living organisms such as hibernatinganimals, seeds and spores do for similar purposes. We began to identifythe likely gene products that the pathogen uses to store TG and torelease the fatty acids for catabolism. We also initiated thedevelopment of an in vitro dormancy model to test the hypothesis thatlipid storage and mobilization are of importance for latency, a modelthat can be adapted for screening antilatency drug candidates.

TG is an important storage form of lipid that accumulates in speciesbelonging to the actinomycetes family, particularly Mtb. IntracellularTG inclusion bodies were detected in mycobacteria isolated from organlesions and Mycobacterium bovis BCG was reported to preferentially useTG within macrophages indicating that TG is probably used as an energysource by Mtb during the course of the disease. We have shown that TGaccumulates when Mtb is subjected to hypoxia or nitric oxide treatmentthat led to a dormancy-like state in culture. We identified fifteenmembers of a novel class of diacylglycerol acyltransferase genes whichwe designated as tgs (triacylglycerol synthase). Several of the tgsgenes were significantly upregulated under hypoxic conditions and undernitric oxide treatment, particularly those that show the highest TGsynthase activity when expressed in E. coli. We identified Rv3130c asthe prime gene in the biosynthesis of TG in the bacterium under in vitrodormancy-like conditions. Our hypothesis was strongly supported by aimportant recent report on the W/Beijing lineage of Mtb strains whichhas been associated with the increasing incidence of multi-drugresistant (MDR) TB epidemic in Asia. The W/Beijing strains were shown tooverproduce TG and the Rv3130c gene was constitutively upregulated alongwith the dormancy regulator protein DosR. The authors suggested thatconstitutive accumulation of TG by this strain may confer an adaptiveadvantage for growth in microaerophilic or anaerobic environments andthus be related to the epidemiological spread of this strain. Ourhypothesis concerning the importance of Rv3130c is strongly supported bythe remarkable finding by our collaborators. A recently developed twostep multiplex and real time PCR method was adapted for reliablequantitative gene profiling of the small amount of latent Mtb expectedto be found in infected animal and human host lung tissues. Remarkably,tgs1 (Rv3130c) was by far the most upregulated gene in the pathogenwithin the host, while dosR and aceAa that are well-known to be involvedin dormancy, were much less induced. Many organisms use waxy esters (WE)as the major form of energy storage. Mtb also stores WE but the genesinvolved in the synthesis of WE and the growth conditions that cause itsaccumulation have not been identified. The basic mechanisms used forbiosynthesis of WE were first elucidated in our laboratory severaldecades ago and the enzymatic strategy described more recently. We haverecently shown that Rv3391 and Rv1543 encode acyl-CoA reductasesinvolved in WE synthesis in Mtb. Rv3391 has been reported to beupregulated under nutrient stress conditions. We found that WEaccumulates under stress conditions that lead to a dormancy-like stateand the accumulated WE is utilized upon starvation. This utilization wasreduced in lipY mutant, indicating the involvement of lipY in WEhydrolysis. Thus, Mtb can produce and use both major energy storageforms. TG and WE, and both forms are likely to be used for successfullygoing through dormancy. WE may also be a component of the cell walllipids that control permeability.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a method of inducing latency in Mycobacterium, the methodcomprising growing a pure culture of Mycobacterium exposed to multiplestress conditions, the stress conditions including at least a lownutrient culture medium without glycerol, a low pH, a relatively highlevel of carbon dioxide and a relatively low gas phase oxygen level.

A latent culture of Mycobacterium growing in vitro is particularlyuseful in evaluating the effectiveness of antimicrobial compoundsagainst this form of the organism, which is prevalent throughout theworld in infected but asymptomatic persons. Before the presentinvention, it was difficult to test drug effectiveness against latentMtb due to the lack of an easily reproducible model system. Accordingly,the present invention discloses an in vitro model of latentmycobacterial infection which is useful in testing antimicrobials foractivity against the infection in its latent stage.

The method of the invention includes growing the Mtb in a low nutrientmedium comprising approximately 10% Dubos medium, preferably at a pH ofapproximately 5 and in an atmosphere relatively high in level of carbondioxide, at approximately 10%. Additionally, the atmosphere includes arelatively low oxygen level of approximately 5%. Preferably, in themethod, the Mycobacterium is a strain of Mycobacterium tuberculosis.

Another embodiment of the present invention includes a method ofinducing a pure culture of Mycobacterium to become rifampicin resistantand to store an increased lipid content, two hallmarks of latency, themethod comprising growing the culture simultaneously exposed to multiplestress conditions, the stress conditions including at least a lownutrient culture medium without glycerol, a low pH, a relatively highlevel of carbon dioxide and a relatively low gas phase oxygen level.

The present invention also includes a pure culture, and even a singleisolated cell of resistant Mycobacterium generated according to themethod disclosed.

The invention includes an in vitro model of latent tuberculosis, themodel comprising an isolated culture of THP1 derived macrophagescontaining ingested Mycobacterium tuberculosis bacteria and incubatedunder hypoxic conditions for a time sufficient for the bacteria toaccumulate increased lipids therein. More broadly, the invention alsoprovides an in vitro model of latent mycobacterial infection, the modelcomprising an isolated culture of THP1 derived macrophages containingingested Mycobacterium spp. cells and incubated under hypoxic conditionsfor a time sufficient for the bacteria to accumulate increased lipidstherein. More broadly still, the invention teaches an in vitro model ofmycobacterial infection, the model comprising an isolated culture ofTHP1 derived macrophages containing ingested Mycobacterium spp. cells.

With regard to the various models disclosed in the invention, theteachings also comprise a method of making a model of latenttuberculosis, the method including inducing cultured THP1 cells todifferentiate into macrophages; infecting the macrophages withMycobacterium tuberculosis bacteria; and incubating the infectedmacrophages under hypoxia, particularly wherein incubating is for a timesufficient for the bacteria to accumulate increased lipids therein, ahallmark of latency. This method is, in general, should also beapplicable to other Mycobacterium species as well.

The various in vitro models of latent tuberculosis and mycobacterialinfection herein disclosed are useful in evaluating compounds foreffectiveness against these bacterial pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1 is a demonstration of the accumulation of storage lipids in Mtbcells treated for the indicated periods under the multiple stressconditions, according to an embodiment of the present invention; TLC wasperformed as described; the plates were charred and quantitation wasdone by densitometry;

FIG. 2 shows increasing lipid storage bodies in Mtb cells withincreasing periods of multiple stress; non-acid fast staining cells(green) and lipid storage body staining (red) increased with time undermultiple stresses; cells were stained with Auramine-O and Nile Red andexamined by confocal laser scanning microscopy (Leica TCS SP5) withZ-stacking to get the depth of the scan field; scanned samples wereanalyzed by LAS AF software for image projection;

FIG. 3 shows an increase in the percentage of lipid-stained cells anddecrease in percentage of acidfast stained cells in Mtb culture whensubjected to multiple stresses in vitro;

FIG. 4 depicts TG accumulation by tgs1 (Rv3130c) and restoration of TGaccumulation by complementation under 18 days of multiple stress; equalamounts of lipid were subjected to TLC as in FIG. 1; C-Δtgs1, is acomplemented mutant;

FIG. 5 shows real-time PCR measurements of transcript levels of tgs andstress responsive genes in Mtb H37Rv under in vitro multiple stress for9 days; comparative C_(T) method (ΔΔC_(T)) was used to quantify andvalues obtained with starting aerated cells were used to calculate thefold induction;

FIG. 6 shows expression profiles of genes encoding proteins involved inthe glyoxylate cycle during the multiple stress treatment;

FIG. 7 depicts a decrease in buoyant density of Mtb cells subjected tomultiple stresses; Mtb cells subjected to the multiple stresses wereplaced on the preformed gradient and centrifuged at 400 g for 20 min;the center tube is a 3 day cell sample mixed with density marker beads;Percoll® gradients were self-formed by centrifugation from a startingsolution with a density of 1.0925 gm/ml; the densities of selected beadlayers (ρ, in gm/ml) are given on the right and the positions of one mlfractions collected for analyses are at the left; numbers below thetubes indicate the number of days under multiple stress:

FIG. 8 is a bar graph showing that Alamar Blue assay reveals developmentof Rif resistance by multiple-stressed Mtb cultures; Mtb culturessubjected to multiple stresses were assayed by the specially adaptedAlamar Blue method described in text for resistance to Rif and INH;fluorescence readings above 0 h controls are depicted;

FIG. 9 shows real time PCR measurement of transcripts levels of a subsetof selected dormancy metabolism and stress responsive genes in Mtb H37Rvunder in vitro multiple stresses for 9 and 18 days; a relativequantitation method (ddCt) was used with the 7500 Fast real time system;samples of starter cultures were used as calibrator to calculate thefold induction:

FIG. 10 are photomicrographs where Oil Red-O staining reveals lipiddroplet accumulation in TDM incubated for 3-days in 1% O₂, 5% CO₂ (a)compared to 0-day control (b);

FIG. 11 indicates the increase in lipid bodies in TDM infected with Mtband subjected to hypoxia for 0 and 3-days;

FIG. 12 shows Mtb within TDM stained with Auramine-O and Nile Redshowing spherical fluorescent lipid bodies and lack of acid faststaining;

FIG. 13 shows that TG accumulated by TDM under hypoxia is utilized byMtb; in A, lipids from uninfected (U) and infected (I) TDM, incubated in20% O₂ or 1% O₂ for 7 days after infection, were resolved on TLC andvisualized under UV light after spraying with 2′,7′-dichlorofluorescein;in B, lipids of Mtb recovered from TDM incubated in 20% O₂ (i) or 1% O₂(ii); solvent was hexane-ether-formic acid (90:10:1, v/v/v) TG,triacylglycerol, FA, fatty acids;

FIG. 14 depicts the fatty acid composition of TG from Mtb recoveredafter TDM infection; after infection with Mtb, TDM were incubated under1% O₂ for 7 days; TG from Mtb isolated from TDM was purified bypreparative TLC. Fatty acid methyl esters were prepared from Mtb TG andanalyzed using a Varian CP-TAP CB column attached to a Varian CP-3900gas chromatograph under a temperature control program;

FIG. 15 depicts transcriptional profiling of genes in Mtb H37Rv frominfected TDM under hypoxia;

FIG. 16 shows that Mtb inside [¹⁴C]acetate-labeled lipid-loadedmacrophages mobilizes host lipids and accumulates TG enriched insaturated fatty acids; in A, AgNO₃-impregnated silica-TLC purified from[¹⁴C]acetate-labeled lipids of infected macrophages (lane 1) and fromMtb recovered from such macrophages (lane 2); solvent system is 1%methanol in chloroform; in B, is shown reversed-phase TLC analysis offatty acids methyl esters of TG from infected macrophages (lane 1) andfrom Mtb recovered from infected macrophages (lane 2); the solventsystem is acetonitrile:methanol:water:acetic acid (30:70:5:1, byvolume); in C. AgNO₃-impregnated silica-TLC of fatty acids methyl estersof TG from infected macrophages (lane 1) and from Mtb recovered frominfected macrophages (lane 2); the solvent system is hexane:diethylether:acetic acid, 94:4:2, v/v/v, (developed twice);

FIG. 17 shows TDM infected with Mtb and incubated under hypoxia appearto fuse together; TDM infected with Mtb at an MOI of 0.1 and incubatedfor 7 days under 1% O₂ were stained with carbolfuschin followed byhematoxylin and eosin (A) or carbolfuschin followed by methylene blue(B); arrows show Mtb; and

FIG. 18 shows Mtb inside TDM that accumulate neutral lipids loseacid-fastness; intact TDM harboring Mtb were fixed with 4%paraformaldehyde overnight and stained with the fluorescent mycolic acidstaining dye Auramine-O (A) followed by the neutral lipid stain Nile Red(B); arrows indicate Mtb that stained strongly for Nile Red but weaklyfor Auramine-O.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Anypublications, patent applications, patents, or other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

With the foregoing in mind, an in vitro dormancy model that can beadapted to drug screening would help to discover antilatency drugcandidates. in vitro models suitable for such screening are urgentlyneeded. A number of different stresses have been applied to Mtb in vitroin an attempt to generate a dormant state and gene expression changeshave been investigated. Most of these models involve single stressfactors such as oxygen depletion, nutrient deprivation, NO treatment andacidic conditions. The gene expression changes during combined nutrientdeprivation and 10% and 0.2% oxygen stress on stationary phase cultureswere investigated. Some of these stress conditions such as prolongednutrient starvation caused Mtb to become highly Rif-resistant butaccumulation of storage lipids was not tested. On the other hand hypoxicconditions we used that caused TG accumulation did not developresistance to 5 μg/ml Rif (unpublished). The NRP-1 condition wasreported to cause resistance to a lower concentration of Rif (1 μg/ml)but lipid accumulation was not tested. We suggest that bothRif-resistance and lipid storage are hallmarks of dormancy. Sinceindividual stress conditions do not allow the pathogen to fully meetthese criteria, we attempted to mimic the in vivo conditions by applyingmultiple stresses thought to be encountered in vivo by Mtb and testedwhether the pathogen would accumulate storage lipids and developRif-resistance. Bacilli within granulomas encounter low oxygen (5%) butnot hypoxia, high CO₂ (10%) concentrations, low nutrient levels andacidic pH. Based on these reports, we used 5% O₂, 10% CO₂, pH 5.0 and10% Dubos medium in a multiple stress in vitro model. Our preliminaryresults show that the combination of the four stress factors leads toaccumulation of storage lipids (TG and WE), development ofRif-resistance and gene expression changes thought to be associated withdormancy. Some of the gene expression changes are similar to those foundin the pathogen from infected lungs of hosts, including primates and ahuman TB patient.

Both Rif-resistance and storage lipid accumulation are associated withdormancy. However, the commonly used in vitro hypoxia model, does notshow both of these characteristics. Therefore we developed a novelmultiple stress model that applies four different stresses that thepathogen is thought to encounter in the host. We grew Mtb cultures inlow pH (pH 5.0), low nutrient (10%) Dubos medium without glycerol, withhigh (10%) CO₂ and low (5%) oxygen gas phase.

Mtb cultures in 10% Dubos (Difco) medium at pH 5.0 at an OD 600 of 0.2were maintained under 5% O₂+10% CO₂+85% N₂ by replacing the air phaseevery other day; oxygen levels did not change significantly during thetwo day period. After monitoring the progressive changes that happenedto the pathogen, we chose to harvest cells at 3, 9 and 18 days undersuch conditions for more detailed studies. These studies includedexamination of storage lipids, antibiotic (Rif and INH)-resistance, geneexpression changes directly relevant to storage lipid synthesis byquantitative real time PCR (qPCR), and gene expression profiles bymicroarray analyses. TLC showed that WE and TG accumulated under themultiple stress conditions reaching near maximal levels by 9 days FIG.1.

The major wax ester was oleyl oleate and the major fatty acids in the TGwere C16 and C18 with less C26 (data not shown). Under these conditionsmore WE accumulated than TG in absolute amounts. Control samples at pH7.0 or pH 5.0 without additional stress showed no increase in storagelipids.

Nile red staining revealed storage lipid accumulation under the multiplestress conditions. It is well known that Mtb cultures contain aheterogeneous population of cells under different physiological states.As the culture was subjected to multiple stress factors we observeddecrease in acid fast staining cells with increasing lipid body stainingcells from a barely detectable level to a significant percentage of thetotal cells by 18 days (FIGS. 2, 3).

Drug resistance was tested by treatment with 5 μg/ml Rif for 5 daysfollowed by serial dilution and plating. By 9 days about 10% of thecells were found to be Rif resistant whereas the starting culturecontained about 0.03% Rif-resistant cells. Rif-resistance increased upto 18 days, sometimes reaching up to 25% at 5 μg/ml Rif. The tgs1(Rv3130c) disruption resulted in the loss of Rif-resistance which wasrestored in the complemented mutant (Table 1). Hypoxic conditions, thatwere previously found to cause accumulation of storage lipids, did notcause the cells to develop detectable Rif-resistance at 5 μg/ml. Thetgs1 (Rv3130c) disruption resulted in loss of TG accumulation undermultiple stress. However the complemented mutant showed a level of TGaccumulation comparable to the wild type (FIG. 4).

TABLE 1 Development of Rif-resistance in wild type H37Rv but not inRv3130c mutant upon application of multiple stress: complementationrestores Rif-resistance. Aliquots were either untreated or treated withRif (5 μg/ml) or INH (0.8 μg/ml). Mtb Resistance to Antibiotics (%)strains Days INH (0.8 μg/ml) Rif (5.0 μg/ml) WT-H37Rv 0 day 0.034(±0.02) 0.037 (±0.027) 9 day  34.7 (±12)  4.7 (±1.9) 18 day   84.4(±17.5)  12.5 (±3.4) Δ-Rv3130c 0 day 0.011 (±0.01) 0.025 (±0.019)(Δ-tgs1) 9 day  21.1 (±7.8)  1.16 (±0.87) 18 day   31.2 (±13.1)  1.89(±0.9) Comple-Δ- 0 day 0.041 (±0.02) 0.029 (±0.01) Rv3130c 9 day  37.9(±13.5)  5.2 (±2.1) 18 day    91 (±19)   11 (±4.5) ND, Not determined;d, day.

Gene expression changes directly relevant to storage lipid accumulationwere examined by real-time PCR. Among all the tgs genes, induction oftgs1 (Rv3130c) was by far the highest at 9 days under the multiplestress condition, followed by Rv3371 and Rv3088 (FIG. 5). Microarrayanalysis also indicated upregulation of Rv3371 under multiple stresscondition (data not shown). Upregulation of Rv3088 probably resultedfrom the low pH as it has been previously reported to be induced underacidic stress. Up-regulation of Rv3371 is noteworthy as it was alsoshown to be up-regulated in human lung granuloma by microarray analysis.The degree of induction of tgs1 (Rv3130c) was comparable to that of icland acr (hspX), genes previously reported to be induced duringpersistence. Our preliminary experimental results raise the possibilitythat lipid accumulation under different stress conditions might usedifferent sets of tgs genes.

The TIGR Pathogen Functional Genomics Resource Center provided the Mtbgenome microarray for this study. Under our multiple stress condition,genes that encode enzymes involved in glyoxylate cycle such asisocitrate lyase (aceA) and citrate synthase (gltA1) showed significantincrease in expression for all time points examined (FIG. 6). From thesedata we infer that the metabolic regulation of cells adapting to themultiple stresses was similar to that observed in persistent bacilliadapting to the phagosomal environment of a macrophage. Under multiplestresses, Mtb showed shutdown of both ATP/NAD energy regenerationsystems. While gene expression for anaerobic respiration wascontinuously increased at the later time points, the aerobic respirationwas significantly repressed at all the time points. All the subunitsencoding NADH dehydrogenase and the ubiquinol-cytochrome C complex wererepressed more than 2-fold. In addition, the expression of the genesencoding ATP synthase subunits was repressed. Slowdown of thetranscription/translation apparatus was evident during the multiplestresses. Many genes related to transcription and translation apparatuswere all consistently repressed. Genes involved in modification ofchromosome and cell division were repressed by the multiple stresses.The expression level of the gene cluster, mas, fad28, mmpL7, and ppsA-E,associated with phthiocerol dimycocerosate (PDIM) synthesis andtransport, that was repressed at the beginning of the multiple stresstreatment, gradually increased more than two-fold and remained highthroughout the period of in vitro multiple stress. Further, the mas-likegene pks2, which is responsible for encoding a hepta/octa-methylbranched fatty acid synthase, was highly expressed. These changes areconsistent with the report that dormant cells have thickened walls. Wealso found significant induction of the genes classified as the stressresponse genes (eg. hspX) that has been suggested to play a role inmaintaining long term survival within the host. The gene array analysisresults were verified by qPCR analysis of selected test genes. Repressedand induced gene transcript level changes indicated by microarrayanalysis were found to be consistent with the changes indicated by theqPCR method (data not shown).

We investigated whether the lipid accumulation that occurs as a resultof multiple stresses might be reflected in changes in buoyant density.We fractionated the culture on a Percoll® gradient in 10 ml SetonEasy-Seal polyallomer centrifuge tubes with Seton Noryl crown assembly.This procedure resolved cells based on buoyant density (FIG. 7). Thebanding pattern changed as the cultures were subjected to multiplestresses for increasing duration. These changes are consistent with theconclusion that application of multiple stresses caused progressivechanges in lipid accumulation resulting in increasing percentages ofcells in the lighter fractions. Auramine-O/Nile Red staining of thedifferent fractions showed that with increasing periods under themultiple stresses, increasing percentage of cells became lipid-loadedand lost acid-fast staining (presumably dormant cells). Staining ofPercoll® fractions from 18 day stressed cultures showed that the lighterfractions were more enriched in, lipid loaded cells that lostacid-fastness. After this long stress period most cells were in thelighter fractions (FIG. 7). Upon Percoll® density gradient fractionationof the 18-day multiple-stressed culture, the great majority of the cellswere distributed in the lighter fractions. When Rif-resistance wasassessed by the Alamar Blue dye method the lighter fractions showed ahigher percentage of Rif-resistant cells (data not shown). The smallnumber of heavier cells present in this culture showedRif-susceptibility comparable to that of the starting culture.

Recent meta-analysis of Mtb microarray data from many in vitro and invivo conditions that are thought to induce dormancy (Murphy and Brown,BMC Infect. Dis. I, 84-100, 2007) indicated that a set of genes possiblyinvolved in lipid storage and utilization are highly upregulated. Wehave determined the transcriptional profile of the genes, selected onthe basis of the meta-analysis by real-time PCR using 7500 Fast system(Applied Biosystem). Detection of transcriptional upregulation of theknown dormancy-responsive genes such as hspx, icl and dosR (FIG. 1) isconsistent with our conclusion that the multiple stress conditionsinduce dormancy. Seven of the 10 genes in the first priority group, suchas Rv3130c along with a few other tgs genes (Rv3371, Rv1760), a fewgenes encoding potential hydrolases (lipX lipY, cut3), and fattyacyl-CoA reductase gene (Rv3391) showed upregulation. Three of the 21genes in the next priority group showed upregulation under the multiplestress condition. One of them showed surprisingly high induction. Theother two upregulated genes in this group were cut2 and lipZ. Only onegene (Rv2285, a tgs) in the third priority group showed upregulation.Among the tgs products that showed the highest enzymatic activity (whenexpressed in E. coli) only this tgs showed a preference for oleoyl-CoA.We already have mutant for this gene and this mutant showed the secondmost impaired ability to accumulate TG under hypoxia as indicated in thepreliminary results presented in the application.

It is noteworthy that 7 of the 10 genes in the first priority group and3 out of 21 genes from the second priority group and only one out of 17in the third priority group showed upregulation. Since theprioritization is based on meta-analysis of the degree of theirupregulation under a variety of conditions thought to induce dormancy,our results suggest that the multiple stress model reflects realdormancy and adds validity to our approach.

The tuberculous granuloma, which is thought to be a hypoxic environment,consists of a core of Mtb-infected macrophages surrounded bylipid-loaded macrophages, mononuclear phagocytes and lymphocytesenclosed by a fibrous cuff. The differentiation of macrophages intolipid-loaded macrophages in tuberculous granulomas is a well-documentedobservation and the secretion of cytokines by the infected lipid-loadedmacrophages probably helps to maintain the granuloma. Histologicalstudies revealed the presence of lipid-loaded macrophages in thegranulomas of immunocompetent and HIV-1 infected patients with TB.Lipid-loaded macrophages contain abundant cytosolic stores of TG andcholesterol esters. A recent study showed that exposure of humanmacrophages to hypoxia (1% O2) converted them into lipid-loaded cellsand M. bovis BCG infection induced the conversion of macrophages intolipid-loaded cells but the nonpathogenic Mycobacterium smegmatis failedto induce lipid body formation. Thus lipid bodies within Mtb-infectedmacrophages may have important roles in pathogenesis and possibly inlatency. Human THP-1 monocytic cell line-derived macrophages (TDM) areknown to be converted into lipid-loaded macrophages. Therefore they canserve as a more convenient experimental model for studies on Mtbmutants, because their use can avoid the variability in responsesencountered in the use of human peripheral blood monocyte-derivedmacrophages and provide a readily available uniformly reproducible cellmodel suitable for high throughput screening of drug candidates. Lipidbodies have been found in Mtb obtained from patients with activedisease. However, the origin of these lipids remains unknown. Thepathogen inside the lipid-loaded macrophages might utilize fatty acidsderived from the lipid bodies in the host cells to store lipids withinMtb for later use. Such a possibility was raised by the recent findingthat adipocytes might be a home for dormant Mtb in humans. In fact, Mtbinside adipocytes were found to accumulate lipid bodies while becomingdormant, as indicated by their resistance to killing by drugs. The lipidbodies found in the pathogen from patients probably originate from thelipid bodies in the macrophages. Our results indicate that Mtb withinlipid-loaded macrophages can use the host's TG to accumulate TG withinthe pathogen and this Mtb becomes Rif resistant meeting our criteria fordormancy.

A New Lipid-Loaded Macrophage Model of Dormancy

Herein we disclose a newly developed THP-1 derived macrophage (TDM)system for infection with Mtb. THP-1 cells, differentiated intomacrophages by treatment with 100 nM PMA for 3 days, were incubated for3 days in 1% O2 and 5% CO2. Oil Red-O staining revealed lipid dropletaccumulation in such macrophages (FIG. 2). When the TDM were infectedwith Mtb at a multiplicity of infection (MOI) of 1.0 for 4 hr andincubated in 1% O₂/5%/CO₂ for 3 days, lipid bodies accumulated in thehost cells (FIG. 3). Mtb cells within the macrophages showed Nile Redstained lipid bodies (FIG. 4). Most Mtb cells showed loss of acid faststaining and thus stained only red; a few showed some acid fast andlipid staining (yellow).

We modified our experimental protocol to allow for longer incubation ofinfected macrophages. We infected TDM with Mtb at an MOI of 0.1 (1bacillus per 10 macrophages) and extended the incubation of the infectedTDM to 7 days under hypoxia. After 7 days, infected TDM were lysed andthe cell debris was removed by centrifugation at 300 g for 10 min. TheMtb cells were pelleted by centrifugation at 3000 g for 10 min andwashed. Lipids from the host-lipid bodies were extracted from thesupernatant and the lipids from the pelleted To analyze fatty acidcomposition, host or Mtb TG was purified by preparative TLC and themethyl esters generated by BF3/methanol transesterification, wereanalyzed by capillary GC. The amount of fatty acids from the TG isolatedfrom Mtb, recovered for TDM, is more than enough for such GC analysis(FIG. 6) pathogens were extracted. TLC analysis of the lipids revealedthat TG in the host cells was markedly increased by incubation underhypoxia for 7 days and the levels of TG were lower in infected TDM underhypoxia (FIG. 5A). Lipids extracted from Mtb recovered from infected TDMwere also analyzed by TLC. We detected increased TG level in Mtb cellsrecovered from TDM incubated under hypoxic conditions (FIG. 5B).

The fatty acid composition of the TG from the pathogen was not identicalto that of the host TG. C16:0, C18:0 and C18:1 fatty acids were thedominant components in both the pathogen and the host. Longer chainsaturated fatty acids (C24. C26 and C28) that were present in thepathogen TG were absent in the host TG. We conclude that the TG thataccumulated in the pathogen probably consisted of fatty acids from thehost and some fatty acids generated within the pathogen.

Gene expression changes occurring in the pathogen within TDM wereexamined using a two-step real time PCR method. Briefly, total RNA wasisolated and purified from a mixture of Mtb-infected TDM using Trizol(Invitrogen) and Qiagen RNeasy column purification method. Total RNA wasDNase treated twice, purified through Qiagen mini elute RNeasy column,purity of RNA was checked at every step. Controls without reversetranscription (RT) verified lack of DNA contamination. First-strandcDNA, synthesized with exo-resistant random hexamers and Superscript IIIreverse transcriptase (Invitrogen) was used for multiplex PCR using manyMtb gene specific primer sets. All primers and Taqman probes weredesigned using VisualOMP6 software from DNA software, Inc (Ann Arbor,Mich.). The Taqman probes have a fluorescein reporter dye (FAM) at5′-end and a Black Hole Quencher (BHQ) at 3′-end. Each multiplex andreal-time PCR primer was checked for specificity and efficiency.Differences in Mtb specific gene transcripts were quantified byreal-time PCR on generated multiplex-PCR products with nested Taqmanprimers and probes. The overall reliability and sensitivity of thetwo-step RT-PCR method to quantify gene expression profiling has beendiscussed in detail elsewhere. We have thus far done only a subset ofgenes thought to be relevant to lipid storage and metabolism (FIG. 7);icl was by far the most induced gene, consistent with the idea that thepathogen in TDM grows on fatty acids. It is noteworthy that lipY, thatwas previously shown by us to be involved in TG mobilization, was highlyinduced and some of the other lip genes also showed induction. dosR andtgs genes were also induced probably indicating their involvement in thestorage of fatty acids derived from host-lipids as TG resynthesizedwithin the pathogen, consistent with our hypothesis. fatp, that might beinvolved in fatty acid transport into the pathogen was also induced.Putative fabp genes also showed some induction. These results indicatethat our hypothesis concerning storage and mobilization of host lipidsby the pathogen has real validity.

We analyzed the resistance of Mtb recovered from TDM after a 7 dayincubation under 20% O₂ or 1% O₂ to Rif and INH by cfu determination.TDM were infected with Mtb at an MOI of 0.1. Mtb cells inside TDM wereexposed to antibiotic for 2 days prior to lysis of TDM and recovery ofthe bacilli. The recovered Mtb cells were diluted and plated on agarplates without antibiotic and incubated for 4 weeks after which cfuswere enumerated. Antibiotic resistance is expressed as percentage ofcontrol without antibiotic. As indicated in Table 2, Mtb recovered fromTDM incubated under 20% O₂ showed resistance to both antibiotics. Othershave found development of Rif resistance in host cells. We found thatRif resistance increased significantly in Mtb recovered from TDMincubated under 1% O₂ for 7 days compared to normoxic conditions. Theseresults indicate support for our hypothesis that lipid-loading ofmacrophages favor the entry of Mtb into dormancy.

Lipid-Loaded Macrophage Model

In making further progress developing the macrophage dormancy model, wetested different MOI in the lipid loaded macrophage system. We assessedthe viability of Mtb-infected lipid-loaded macrophages under hypoxiaunder different MOI. At MOI 1.0 or higher the host cell viability wasseriously compromised. At MOI 0.1, after 7 days under 1% O₂, 40% of theoriginal TDM population remained intact as an adhered monolayer and wereloaded with lipid droplets. About 94% of these lipid-loaded TDM cells inthe adhered monolayer were viable. These results support the notion thatthese lipid-loaded TDMs provide a TG-enriched sanctuary for Mtb,favoring its entry into dormancy.

TABLE 2 Increase in resistance of Mtb inside lipid-loaded macrophages toRif and INH. Mtb within TDM incubated for 7 days under 20% O₂ or 1% O₂was exposed to antibiotic for 2 days. Mtb cells were then recovered bylysis of TDM and plated on agar plates for cfu determination. Mtbrecovered from Resistance to Antibiotic TDM incubated Rif INH 7 days in1 μg/ml 5 μg/ml 0.1 μg/ml 0.8 μg/ml 20% O₂ 9% 4%  25% 12%  1% O₂ 68 ±14% 25 ± 2% 100% 68 ± 18%

We originally suspected that Mtb utilizes the macrophage lipid bodies toacquire fatty acids and store them as TG within the pathogen to enableit to go through dormancy. To test this hypothesis, we labeled TDMlipids by incubating the cells with [¹⁴C]acetic acid or [¹⁴C]oleic acid,under 1% O₂ for 2 days. These cells were washed three times with sterilephosphate-buffered saline (PBS) to remove unincorporated radiolabel.Thin-layer chromatographic (TLC) analysis of the labeled lipidsextracted from TDM showed that major part (about 60%) of theradioactivity in the lipids derived from labeled acetate and oleate wasin TG that accumulated in TDM under 1% O₂. These pre-labeled cells wereinfected with Mtb at an MOI of 0.1 for 4 h under 1% O₂. Followinginfection, extracellular Mtb were removed by thoroughly washing the TDMmonolayer with sterile PBS. Infected TDM were incubated for 5 more daysunder 1% O₂. The host lipids and lipids from Mtb recovered from the hostcells were obtained and the lipids were analyzed by TLC. The fatty acidcomposition of the Mtb and host TG was analyzed by resolving the intactTG and fatty acid methyl esters derived from TG on reversed-phasesilica-TLC and argentation-TLC. Analysis of intact TG from [¹⁴C]acetatein TDM was composed of saturated and unsaturated fatty acids. However,the TG of Mtb was predominantly composed of saturated fatty acids asindicated by greater mobility on AgNO₃-impregnated TLC (FIG. 8 A).Analysis of fatty acid methyl esters prepared from TG isolated from TDMand Mtb indicated that the TG of Mtb, recovered from TDM labeled with[¹⁴C]acetate, was composed primarily of saturated fatty acids, mostly¹⁴C-16:0 and a very small quantity of ¹⁴C-18:0 fatty acids (FIG. 8 B,C).See also Table 3, below.

TABLE 3 Accumulation of radiolabeled TG by Mtb and mutants inlipid-loaded macrophages under hypoxia: DPM in TG % of WT ¹⁴C-Acetate WT210,000 100% ΔRv3130c 22,600  11% ¹⁴C-Oleate WT 29,000 100% ΔRv3130c1470  5%

Mtb recovered from [¹⁴C]oleic acid-labeled TDM had TG that wasdistinctly different in fatty acid composition from the TG in TDM. Whilethe ¹⁴C in TDM TG was predominantly in 18:0 (about 81% of total fattyacids), ¹⁴C in Mtb TG was mainly in unsaturated fatty acids (about 70%of total fatty acids). The identity of these fatty acids is to bedetermined. These results clearly indicate that Mtb acquires fatty acidsfrom TDM lipid bodies for synthesizing TG as a potential energy source.The TG stored within the pathogen probably includes the fatty acids fromthe host lipids and fatty acids generated by modification and/orcatabolism and resynthesis. The biochemical processes involved can bededuced only after further characterization of the TG that accumulatesin the host and in the pathogen.

We also postulated that the Mtb tgs gene products may be pivotallyinvolved in synthesizing TG within the Mtb cell from fatty acidsacquired from host TG. To test this hypothesis, we infected thepre-labeled TDM with wild-type Mtb and tgs1 (Rv3130c) deletion mutant(ΔRv3130c) as described in the methods section. About 1% of theradiolabel in the TG in TDM was found in the TG isolated from Mtb. Wequantitated the radioactivity in the TG of Mtb and Δv3130c recoveredfrom TDM. The results indicate that TG accumulation by the Δ3130c wasdecreased by 90-95% when compared to the wild-type (Table 3). Theseresults additionally suggest that Rv3130c plays an essential role in theaccumulation of TG by Mtb within lipid-loaded macrophages.

In order to visualize the Mtb inside lipid-loaded host cells, infectedTDM after 7 days under hypoxia were fixed with 4% paraformaldehyde andstained for Mtb with carbolfuschin followed by methylene blue orhematoxylin and eosin to stain the host cell. Mtb inside TDM were alsostained with the mycolic acid-specific fluorescent dye Auramine-Ofollowed by Nile Red which stains neutral lipids. Interestingly, as seenin FIG. 9, the Mtb-infected TDM which were incubated under hypoxia for 7days appeared to be fusing together. It is probable that these TDM arein the process of forming multinucleate giant cells (asterisk in FIG.9A) which are known to be present in the hypoxic environment of thegranuloma in close vicinity to lipid-loaded macrophages. Theseobservations support the hypothesis that this lipid-loaded macrophagesystem is a good model for in vivo latency.

Individual Mtb cells inside TDM that accumulated neutral lipids, asindicated by Nile Red staining, lost acid-fastness as shown by weak ortotal loss of Auramine-O staining (FIG. 10). Conversely, Mtb cells whichstained strongly with Auramine-O did not accumulate neutral lipids. Thisaccumulation of neutral lipids and loss of acid-fastness by a subset ofMtb cells within lipid-loaded TDM under hypoxia, correlates well withour data in the Preliminary Results, that demonstrated the developmentof Rif resistance by about 25% of the Mtb population and increase in TGwithin Mtb from TDM under the same conditions. Since then, we havereconfirmed these results with additional experiments.

Based on these results, the multiple stress in vitro latency modeldisclosed herein appears to be the best one available for screeningchemicals to discover drug candidates that can eliminate latentpathogen. Accordingly, in the drawings and specification there have beendisclosed typical preferred embodiments of the invention and althoughspecific terms may have been employed, the terms are used in adescriptive sense only and not for purposes of limitation. The inventionhas been described in considerable detail with specific reference tothese illustrated embodiments. It will be apparent, however, thatvarious modifications and changes can be made within the spirit andscope of the invention as described in the foregoing specification andas defined in the appended claims.

1. A method of inducing latency in Mycobacterium, the method comprisinggrowing a pure culture of Mycobacterium exposed to a plurality of stressconditions, the stress conditions selected from a low nutrient culturemedium without glycerol, a low pH, a relatively high level of carbondioxide, a relatively low gas phase oxygen level, and combinationsthereof.
 2. The method of claim 1, wherein the low nutrient mediumcomprises approximately 10% Dubos medium.
 3. The method of claim 1,wherein the low pH is approximately
 5. 4. The method of claim 1, whereinthe relatively high level of carbon dioxide is approximately 10%.
 5. Themethod of claim 1, wherein the relatively low oxygen level isapproximately 5% in the gas phase.
 6. The method of claim 1, wherein theMycobacterium is a strain of Mycobacterium tuberculosis. 7-22.(canceled)